JP2911956B2 - 5,11-Dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepin-6-one and -thione and methods of using such compounds in the prevention or treatment of AIDS - Google Patents

Abstract

Disclosed are novel 5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-ones and -thiones. These compounds, as well as certain known 5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-ones are useful in the treatment of AIDS, ARC and related disorders associated with HIV infection.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the treatment of HIV infection, including the known and novel 5,11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepin-6-one and -thione. The present invention relates to a pharmaceutical composition for prophylactic treatment.

Acquired immunodeficiency syndrome (AIDS), a human disease,
It is caused by the human immunodeficiency virus (HIV), particularly the virus known as HIV-1.

Like other viruses, replication of HIV-1 requires the biosynthetic machinery of the host cell to be infected. This means that the device synthesizes the structural proteins that make up the progeny of the virus. These proteins are encoded by the viral particles of the infecting virus, the genetic material contained within the virions. However, because HIV is a retrovirus, its genetic material is RNA, not DNA as in the genome of the host cell. Thus, viral RNA must first be transcribed into DNA and then integrated into the host cell's gelom in order for the host cell to produce the required viral proteins.

Transcription from RNA to DNA is performed using reverse transcriptase (RT) contained with the RNA in the infected virion. Reverse transcriptase has three enzymatic functions. That is, as an RNA-dependent DNA polymerase, as a ribonuclease,
Acts as an NA polymerase. First, RT is RNA-dependent DNA
Acts as a polymerase to produce single-stranded DNA using viral RNA as a template. Next, RT acts as a ribonuclease to convert the as-produced DNA into the original viral R
Release from NA and destroy original RNA. Finally, RT is D
Acting as an NA-dependent DNA polymerase, using the first DNA strand as a template to create a second complementary DNA strand. The two strands form double-stranded DNA, a type of DNA found in the host cell genome, and are integrated into the host cell genome by another enzyme called integrase.

Compounds that inhibit the enzyme function of HIV-1 reverse transcriptase inhibit HIV-1 replication in infected cells. Such compounds are useful for preventing and treating HIV-1 infection in humans.

First, the present invention provides a method for treating a human exposed or infected with the HIV-1 virus in a prophylactically or therapeutically effective amount of 5,11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepine. -6
The present invention relates to a method for preventing or treating HIV-1 infection, which comprises administering -on and -thione. These compounds may be new or known. All the compounds are HI
V-1 has an inhibitory effect on RT. Second, the present invention relates to a novel 5,11-dihydro-6H-pyrido [2,3-b]
[1,4] benzodiazepine-6-thione. Third, the present invention relates to a method for producing the novel compound. Fourth, the present invention includes a pharmacological composition suitable for the prevention or treatment of HIV-1 infection, comprising both the novel compound and the known compound described above.

First, the present invention provides a method for treating a human exposed or infected with HIV-1 with a prophylactically or therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable acid addition salt of the compound. The present invention relates to a method for preventing or treating HIV-1 infection, which comprises administering.

(In the formula, Z represents an oxygen atom or a sulfur atom, and R ¹ is a hydrogen atom, an alkyl group or a fluoroalkyl group having 1 to 4 carbon atoms, a cyclopropyl group, an alkenyl group or an alkynyl group having 3 to 4 carbon atoms. ,
2-halo-propen-1-yl group, arylmethyl group (wherein, the aryl moiety represents a phenyl group or a thienyl group, which is terminally substituted or substituted with a methyl group, a methoxy group or a halogen atom), acetyl R ² represents an alkoxyalkyl group or an alkylthioalkyl group having 2 to 3 carbon atoms, R ² represents an alkyl group or a fluoroalkyl group having 1 to 4 carbon atoms, a cycloalkyl group having 3 to 5 carbon atoms, a carbon atom An alkenyl or alkynyl group having 2 to 4 carbon atoms, an alkoxyalkyl group or an alkylthioalkyl group having 2 to 3 carbon atoms, an alkanoyl group having 2 to 3 carbon atoms, a hydroxyalkyl group having 2 to 4 carbon atoms, an arylmethyl group (Wherein the aryl moiety is a phenyl, thienyl or furanyl group) Represents
These are unsubstituted or substituted by an alkyl or alkoxy group having 1 to 3 carbon atoms, a hydroxyl group or a halogen atom), a phenyl group (unsubstituted or an alkyl or alkoxy group having 1 to 3 carbon atoms, a halogen atom) Or an alkoxy moiety represents an alkoxycarbonylmethyl group having 1 to 5 carbon atoms, wherein R ³ , R ⁴ and R ⁵ each independently represent a hydrogen atom or a carbon atom having 1 to ⁵ carbon atoms.
3 to 3 alkyl groups, wherein at least one of these substituents represents a hydrogen atom, or one of R ³ , R ⁴ and R ⁵ is a butyl group, having 2 to 4 carbon atoms.
An alkanoyl group, an alkoxycarbonyl group having 2 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, an alkoxycarbonylalkyl group having an alkoxy portion and an alkyl portion each having 1 to 2 carbon atoms, a halogen atom, Trihalomethyl group, hydroxyl group, alkoxy group having 1 to 3 carbon atoms, alkylthio group having 1 to 3 carbon atoms, alkanoyloxy group having 2 to 3 carbon atoms, alkanoylamino group having 1 to 3 carbon atoms, carbon atom An aminoalkyl group having 1 to 3 carbon atoms, a mono- or di-alkylamino group in which each alkyl moiety contains 1 to 2 carbon atoms, a carboxyalkyl group having 2 to 3 carbon atoms, a cyano group, a nitro group, a carboxy group Group, carbamyl group, amino group, azide group, each alkyl moiety is 1 Mono having 2 carbon atoms stone -
Or a di-alkylaminoalkyl group, provided that the remaining two substituents represent a hydrogen atom or a methyl group,
Or when Z represents an oxygen atom, one of R ³ , R ⁴ and R ⁵ represents an alkylsulfinyl group or an alkylsulfonyl group having 1 to 3 carbon atoms, provided that the remaining two substituents are a hydrogen atom or R ⁶ , R ⁷ , R ⁸ and R ⁹ each represent a hydrogen atom, or one of R ⁶ , R ⁷ , R ⁸ and R ⁹ represents an alkyl group having 1 to 4 carbon atoms An alkanoyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, an alkoxy moiety wherein the alkoxy moiety and the alkyl moiety each have 1 to 2 carbon atoms. Carbonylalkyl group, halogen atom,
A trihalomethyl group, a hydroxyl group, an alkoxy group having 1 to 3 carbon atoms, an alkylthio group having 1 to 3 carbon atoms,
An alkanoyloxy group having 2 to 3 carbon atoms, an alkanoylamino group having 1 to 3 carbon atoms, 1 carbon atom
A mono- or di-alkylamino group wherein each alkyl moiety has 1 to 2 carbon atoms, a carboxyalkyl group having 2 to 3 carbon atoms, a cyano group, a nitro group, a carboxy group, A carbamyl group, an amino group, an azido group, an alkyl moiety represents a mono- or di-alkylaminoalkyl group each having 1 to 2 carbon atoms, and the remaining three substituents represent a hydrogen atom; (Of the three substituents, two represents a hydrogen atom and one represents a methyl group, an ethyl group or a halogen atom.) Second, in the above formula I, Z represents an oxygen atom or a sulfur atom; ¹ is a hydrogen atom, an alkyl group having 1 to 3 carbon atoms,
Represents an allyl group, a propargyl group, a 2-halopropen-1-yl group, a methoxymethyl group or a methylthiomethyl group, wherein R ² is an alkyl group having 1 to 4 carbon atoms, a cycloalkyl group having 3 to 4 carbon atoms, 2 to 4 carbon atoms
An alkenyl group or an alkynyl group, an alkoxyalkyl group or an alkylthioalkyl group having 2 to 3 carbon atoms, an alkanoyl group having 2 to 3 carbon atoms, a hydroxyalkyl group having 2 to 4 carbon atoms, an arylmethyl group (wherein The aryl moiety represents a phenyl group or a thienyl group, which are unsubstituted or substituted by a methyl group, a methoxy group or a halogen atom) or an alkoxycarbonylmethyl group in which the alkoxy moiety has 1 to 4 carbon atoms; R ³ , R ⁴ and R ⁵ each independently represent a hydrogen atom or a methyl group, provided that at least one of these substituents represents a hydrogen atom, or R ⁵ represents an ethyl group, a propyl group or a butyl group, the remaining two substituents represent a hydrogen atom, R ⁶ represents a hydrogen atom, a methyl group or an ethyl group, provided that If R ⁷ represents a hydrogen atom or a methyl group, to R ⁷ is C 1 -C 2 alkyl group, an acetyl group, a hydroxyalkyl group having 2 C 1 -C, alkoxycarbonyl group 3 2 -C, An alkoxycarbonylalkyl group having an alkoxy moiety and an alkyl moiety each having 1 to 2 carbon atoms, a halogen atom, a trifluoromethyl group, a hydroxyl group, an alkoxy group having 1 to 2 carbon atoms, an alkylthio group having 1 to 2 carbon atoms An acetyloxy group, an alkanoylamino group having 1 to 2 carbon atoms or a cyano group, provided that R ⁸ represents a hydrogen atom: R ⁸ represents an alkyl group having 1 to 2 carbon atoms, an acetyl group, A hydroxyalkyl group having 1 to 2 atoms, an alkoxycarbonyl group having 2 to 3 carbon atoms, an alkoxy moiety, and An alkoxycarbonylalkyl group having an alkyl moiety having 1 to 2 carbon atoms each, a halogen atom, a trifluoromethyl group, a hydroxyl group, an alkoxy group having 1 to 2 carbon atoms, an alkylthio group having 1 to 2 carbon atoms, and an acetyl group Represents an alkanoylamino group having 1 to 2 carbon atoms or a cyano group, provided that R ⁷
Represents a hydrogen atom: or R ⁷ and R ⁸ both represent a hydrogen atom, a methyl group or a halogen atom, and R ⁹ represents a hydrogen atom or a methyl group, provided that
R ⁸ relates to the above method wherein a hydrogen atom or a methyl group is represented.

Further, in the present invention, in the above formula I, Z represents an oxygen atom or a sulfur atom, and R ¹ is a hydrogen atom, a 2-halo-2-propen-1-yl group, or an alkyl group having 1 to 3 carbon atoms. Wherein R ² represents an alkyl group having 1 to 4 carbon atoms or a cycloalkyl group having 3 to 4 carbon atoms, and R ^{3 to} R ⁹ each represent a hydrogen atom.

The compounds of the formula I may, if desired, be prepared in a customary manner, for example by dissolving the compound of the formula I in a suitable solvent and acidifying the solution with one or more molar equivalents of the desired acid, to give a non-toxic pharmacological May be converted to a more tolerable acid addition salt. The present invention also includes the salt. The salt is preferably formed at any of R ^{3 to} R ^{9 when} it is a normal amine group.

Examples of inorganic or organic acids which can form non-toxic pharmacologically acceptable acid addition salts with the compounds of the formula I are given below: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, sulfuric acid, tartaric acid ,
Citric acid, methanesulfonic acid, etc. The compounds of the formula I usually form addition salts with one molar equivalent of the acid.

The above desired compounds of formula I inhibit the HIV-1 reverse transcriptase, and thereby inhibit the replication of HIV-1, and are therefore useful in the methods of the present invention.

To carry out the method, the compound of formula I is
Or it is orally administered, parenterally administered, or topically administered in a plurality of times. Suitable oral doses of the compound may range from about 10-500 mg daily. For parenteral preparations, suitable dosage units may contain from 1 to 50 mg of the compound; for topical administration, preparations containing from 0.01 to 1% of the active ingredient are preferred. However, it is to be understood that dosages will vary from patient to patient, and that individual patient doses will vary at the discretion of the physician, and that the physician will use the size, condition, As well as the patient's response to the drug.

When the compound is administered by the oral route, the compound
The compound can be administered as a medicament in the form of a therapeutic medicament containing the compound together with a biocompatible pharmaceutical carrier material. Such a carrier material can be an inert organic or inorganic carrier material suitable for oral administration. Examples of such carrier materials include:
Examples include water, gelatin, talc, starch, magnesium stearate, gum arabic, vegetable oil, polyalkylene glycol, petroleum jelly, and the like. Therapeutic agents are manufactured in conventional manner, and the final dosage form is a solid dosage form, such as a tablet, dragee, capsule, or the like, or a liquid dosage form.
For example, it can be a solution, suspension, emulsion, and the like. Therapeutic agents are subjected to conventional formulation processing, for example, disinfection. In addition, therapeutic excipients may contain the customary auxiliaries, such as preservatives, stabilizers, emulsifiers, flavor improvers, wetting agents, buffers, various salts for changing the osmotic pressure and the like. Solid carrier materials which can be used also include, for example, starch, lactose, mannitol, methylcellulose, microcrystalline cellulose, talc, silica, dibasic calcium phosphate and high molecular weight polymers such as polyethylene glycol.

For parenteral use, the compounds are preferably administered as an aqueous or non-aqueous solution, or as a suspension or emulsion in a pharmaceutically acceptable oil or liquid mixture, which may include antibacterial, oxidizing agents. Inhibitors, preservatives, buffers or other solutes to render the solution isotonic with blood, thickeners, suspending agents or other pharmaceutically acceptable carriers can be included.
Additives of this type include, for example, tartrate, citrate and acetate buffers, ethanol, propylene glycol, polyethylene glycol, complexing agents (eg, EDT
A), antioxidants (eg, sodium bisulfite, sodium metabisulfite and ascorbic acid), high molecular weight polymers for viscosity control (eg, liquid polyethylene oxide), and polyethylene derivatives of sorbitol anhydride. If necessary, preservatives, for example benzoic acid, methyl or propylparaben, benzalkonium chloride and other quaternary ammonium compounds may be added.

The compounds of the present invention may also be administered as inhalable solutions, which may contain, in addition to the compounds of the present invention, suitable buffers, stability modifiers, antimicrobial agents, antioxidants and viscosity enhancers in aqueous solvents. it can. Examples of additives used for increasing the viscosity are polyvinyl alcohol, cellulose derivatives, polyvinylpyrrolidone, polysorbates or glycerin. The antimicrobial agent used can include benzaconilum chloride, thimerosal, chlorobutanol or phenylethyl alcohol.

In addition, the compounds can be administered as suppositories.

With respect to the composition, the present invention relates to a novel compound wherein R ^{1 to} R ⁹ have the meanings as defined in the above formula I and Z represents sulfur, and a pharmaceutically acceptable acid addition salt of the compound. including.

The present invention further includes novel compounds wherein Z is an oxygen atom in the above formula I, as well as pharmaceutically acceptable acid addition salts of the compounds. Examples are the following compounds: a) 5- (2-chloro-2-propenyl) -11-ethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepine-6 B) 2,5,8,11-tetramethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepin-6-one; c) 5,11-diethyl -10-methyl-5,11-dihydro-6H
-Pyrido [2,3-b] [1,4] benzodiazepin-6-one; d) 5,8-dimethyl-11-ethyl-5,11-dihydro-6H
-Pyrido [2,3-b] [1,4] benzodiazepin-6-one; e) 2,5,10,11-tetramethyl-5,11-dihydro-6H-
Pyrido [2,3-b] [1,4] benzodiazepin-6-one; f) [11- [5,11-dihydro-6-oxo-6H-pyrido [2,3-b] [1,4] Benzodiazepin-11-yl]]-
Tert-butyl acetate; g) 2-chloro-5-ethyl-11-methyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepin-6-one; and h) 5,8,11-Trimethyl-5,11-dihydro-6H-pyrido1 [2,3-b] [1,4] benzodiazepin-6-one.

In the above formula I, the compound in which Z represents an oxygen atom can be produced by the following general methods A, B and C.

Method A In the method of the present invention, a compound represented by the following formula is cyclized.

Wherein Ha ¹ represents a halogen atom and R ^{1 to} R ⁹ have the meanings as defined above. Cyclization involves bringing the compound of formula II to an elevated temperature, preferably 150 ° C. or higher, advantageously trichlorobenzene,
In the presence of a high boiling inert solvent such as paraffin oil or sulfolane, in the presence of a basic catalyst such as potassium salt or copper powder, but preferably in the presence of an acid catalyst such as hydrogen chloride or concentrated sulfuric acid. It can be performed by heating. formula
When the II compound is heated, the generation of hydrogen halide starts at a temperature of 150 ° C. and stops after a few hours of heating. Thereafter, high-boiling solvents which may be present are distilled off under reduced pressure and, upon cooling, the desired end product of the formula I crystallizes. Thereafter, the assembled product is purified by a conventional method, for example, recrystallization from an inert solvent.

Method B: In the present invention, the following formula: (Wherein R ² and R ⁶ represent the meanings defined above) by the following formula: Wherein R ¹ and R ^{3 to} R ⁵ have the meanings defined above, and Ha 1 represents a halogen atom.
React with 3-aminopyridone. The reaction is carried out at a temperature above 150 ° C. The process allows the compounds of the formula I to be prepared in good yields by a continuous reaction process. The reaction may be carried out without using a solvent, or may be carried out in the presence of a high boiling point inert solvent such as trichlorobenzene, tetraethylene glycol, tetrahydronaphthalene, sulfolane, etc., and a catalytic amount of a mineral acid such as hydrogen chloride or It can be performed in the presence of concentrated sulfuric acid. When the reaction mixture is heated, carbon dioxide is first evolved at a temperature between 120 ° C. and 150 ° C., forming the compound of formula II above as an intermediate. However, this intermediate does not need to be isolated and, in situ, the reaction mixture containing it is brought to 150 ° C or higher, preferably 180-250 ° C.
Cyclization accompanied by generation of hydrogen halide. Of course, the reaction mixture of the compounds of formulas III and IV can also be heated to the temperature required for direct cyclization.

Method C: In the above example of producing a compound wherein R ¹ represents a hydrogen atom according to Method A or B according to Method I or B, if desired, such a compound may be converted to a compound of the formula I wherein R ¹ It can also be converted to a compound representing a hydrogen atom. To do this, the compound in which R ^{1 in} formula I above represents a hydrogen atom is first reacted with an alkali metal hydroxide, alkali metal alcoholate, alkali metal amide or alkali metal hydride to form a 5-alkali metal The compound is formed as an intermediate. The resulting 5-alkali metal compound is then represented by the following formula: R ¹ Y (where R ¹ represents a hydrogen atom other than the above defined meanings, Y represents a chlorine atom, a bromine atom, (Representing an iodine atom, an anion of a reactive ester, or a leaving group such as a sulfate or a residue of an aliphatic or aromatic sulfonic ester)).

A method for preparing the starting compound of Formula II is described in U.S. Pat.
No. 4, and 3,406,168. All starting compounds of the formulas III and IV are known or can be prepared by methods described in the literature.

As mentioned above, the compounds provided by the present invention are HIV
-1 suppresses the enzyme activity of RT. Testing these compounds allows them to be tested for HIV RT, as shown below.
It can be seen that the RNA-dependent DNA polymerase activity is inhibited. Other tests not described here believe that they also suppress the DNA-dependent DNA polymerase activity of HIV RT.

Using the reverse enzyme (RT) assay described below, HIV
One can test the inhibitory effect of RT on RNA-dependent DNA polymerase activity. The specific compounds described in the examples below were tested.
Table I shows the results of this test.

Reverse transcriptase (RT) analysis Analytical logic: The enzyme encoded by the human immunodeficiency virus (HIV-1), which transcribes DNA from the RAN template, includes an enzyme called reverse transcriptase (1). This activity has been described in the literature, and the reverse transcriptase was synthesized by a synthetic template [oligo d (G)
Use poly r (C)] starting with, as a substrate ³ H-
It can be quantitatively measured by cell-free enzyme analysis (2) based on the viewpoint that transcription for synthesizing a radiolabeled acid-precipitable DNA chain using dGTP can be performed.

Materials: a) Manufacture of enzyme A reverse transcriptase from the LAV species of human immunodeficiency virus (HIV-1) (1) was prepared by using the DNA clone pBRTprt1 + (2) under the control of the 1ac promoter of the expression vector pIBI21 (4).
Was isolated from the strain JM109 (3) expressing Positive selection (p
overnight culture in 2XYT medium (37 ° C., 225 rpm) (5) supplemented with 100 μg / ml ampicillin for ositive selection), at a dilution of 1:40, 10 μg / ml thiamine,
0.5% casamino acid and 50 μg / ml ampicillin (5)
To M9 medium supplemented with. Culture the OD540 to 0.3-0.4
(37 ° C, 225 rpm). 0.5 mM repressor inhibitor IPTG (isopropyl bD-thiogalactopyranoside) is added and the mixture is incubated for a further 2 hours. Pellet bacteria, 50 mM Tris, 0.6 mM
EDTA, resuspended in 0.375 M NaCl buffer and digested by adding lysozyme (1 mg / ml) on ice for 30 minutes. Cells are lysed by adding 0.2% NP-40 and transferred into 1 M NaCl.

After removal of insoluble debris by centrifugation, the protein is precipitated by adding 3 volumes of a saturated aqueous solution of ammonium sulfate. Pellet the enzyme and add RT buffer (50 mM Tris pH 7.5, 1 mM EDTA, 5 mM DT
T, 0.1% NP-40, 0.1 M NaCl, and 50% glycerol) and store at -70 ° C for further use.

b) 2-fold diluted Nonidet P-40 0.1% 1M of MgCl 4mM NaCl 120mM 1% of dithiothreitol 40 mM 1M of Tris pH 7.4 100 mM 1M concentration 1M composition bacteria reagent twice mixture of bacteria reaction mixture [poly r (C) / oligo d (G)] (5: 1) 2 μg / ml ³ H-dGTP (81 μM) 0.6 μM Analysis method: A 2-fold diluted bacterial reaction mixture is collected and stored at −20 ° C. The mixture is stable and is dissolved for use in each analysis. This enzyme assay has been applied to a 96-well microtiter plate system and has been described in the literature (6). Tris buffer (50 mM, pH 7.5), vehicle (diluted with solvent to match compound dilution), or compound in vehicle, can be added to a 96-well microtiter plate (10 μ /
(3 holes / compound). HIV RT enzyme 50mM T
ris pH 7.4, diluted and diluted, containing 15 μl of the diluted enzyme containing 0.001 units (1 unit is the amount of enzyme capable of transforming 1 μM substrate per minute at 25 ° C.) into one well Allow 15μ to be dispensed. 20μ 0.12-0.5M EDTA in the first three holes of the microtiter plate
Is added. EDTA chelates to existing Mg ²⁺ and prevents reverse transcription. This group is used as background for samples drawn from all other groups. Place 25μ of the 2x diluted reaction mixture in all wells,
Incubate the analyte at room temperature for 60 minutes. The analysis is terminated by precipitating the DNA in each well with 50 μ of trichloroacetic acid (TCA) (10% w / v) in sodium pyrophosphate (1% w / v). Microtiter plate at 4 ° C
And precipitates on 30 glass fiber paper (Schleicher & Schuell) on a Skatron semi-automatic harves.
ter). Thereafter, the filter is further subjected to TCA (5%) containing sodium pyrophosphate (1%).
And rinse with aqueous ethanol (70%), dry and then transfer the scintillation vial (6). Each vial contains 2 ml of scintillation cocktail,
Multiplied by the Beckman beta counter. The inhibition rate (%) is calculated by the following formula: References: 1. Benn, S., et al., SCIENCE 230: 949, 1985 2. Farmerie, WGet al., SCIENCE 236: 305, 1987 3. Yanisch-Perron. C., Viera, J., and Messing , J., GENE
33: 103,1985 4.International Biotechnologies, Inc., New Haven, CT
06535 5.Maniatis, T, Fritsch, EF, and J.Sambrook, eds.MOLEC
ULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbo
r Laboratory, 1982 6.Spira, T., et.al.J.Clinical Microbiology, 25: 97,198
7 The compound effective for RT analysis is HIV-1
To confirm that it has the ability to suppress replication,
Some of the compounds of formula I are tested by the human T cell culture analysis shown below.

Human T cell culture analysis Theory of analysis: The formation of syncytia is HIV-1
Are characteristics of in vitro cultures of infected CD4 + T cells. In this analysis, T cells are treated with a putative replication-suppressing compound and subsequently infected with HIV-1. After the culture, the culture is examined for the formation of syncytia.

Analytical method: The target cell, designated c8166, is a subclone of human lymphocyte cells of T cell origin, on a 96-well flat bottom plate, RPMI 1640 (+ 10% fetal bovine serum) medium 100
Make an initial concentration of 5 × 10 ⁴ per μ. It contains a selected amount of the test compound dissolved in DMSO. 24 hours later, 50-100 TCID _50'S ( ₅₀ % of test culture)
% Of HIV-1 (2) HTLV-I
Inoculate each medium of the II B strain. Control medium contains either compound or virus only. Four days after virus inoculation, cultures are observed for the number and variance of virus-induced giant cell syncytia. The inhibition rate (%) of the test compound is determined by comparing with the control powder. The presence or absence of virus replication is confirmed by examining the presence or absence of infectious progeny due to the induction of syncytial formation in the second T cell culture three days after harvesting the cell-free culture from the experimental glue.

References: (1) M. Somasundaran and HL Robinson, Science 24
2 , 1544 (1988) (2) GMShaw, RHHahn, SKArya, JEGroopman, R.
C. Gallo and F. Wong-Staal, Science 226 , 1165 (1984) In order to evaluate the characteristics of the compounds provided by the present invention for their enzyme inhibitory activity, some were analyzed using Feline analysis methods known per se. Leukemia Virus-induced reverse transcriptase and bovine thymus-derived DNA were tested for their ability to inhibit α-polymerase. None of the tested compounds had an inhibitory effect on these enzymes. These results indicate that the enzyme-suppressing activity of the compounds provided by the present invention can be said to be rather specific for HIV RT.

To roughly determine the cytotoxicity of the compounds provided by the present invention, some compounds are tested by the following MTT cytotoxicity assay. The results of the test are reported in Table 1. Compounds having a relatively high EC ₅₀ of preferred.

MTT assay for cytotoxicity Analysis theory: MTT [3- (4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide)
It is based on the cleavage of tetrazolium bromide by metabolically active cells, which results in a very strong blue coloration. This analysis is described in the literature (1) but is optimized for the test purposes reported here.

Analysis method: H9 cell line (2), that is, 10% fetal bovine serum was added.
An established human lymphocyte suspension cell line cultured in RPMI 1640 is used as the target cell line in the assay. Cells (10
0μ) is placed in the wells of a microtiter plate at a concentration of 10 ⁵ cells / ml in the presence of various concentrations of inhibitor. The cells are cultured at 37 ° C. in a humidified CO ₂ incubator. Five days later, 20 μM MTT (PRMI1640
5 mg / ml, sonicated, 0.2 μm filtered and stored at 4 ° C.) is added to each well. Plus 37
After culturing at 4 ° C. for 4 hours, add 60 μl of Triton-X to each well and mix thoroughly to dissolve the crystals. Absolute ethanol (5μ) was added to each well, and the resulting mixture was incubated at 60 ° C for 30 minutes and read on a plate reader (Dynatech).
Read immediately at a wavelength of 570 nm using.

Non-glandular regression analysis was performed using the data from this analysis, and EC
₅₀ , ie the highest dispersion of non-toxic concentrations.

References: (1) Mosmann, Tim, J. Immunol. Methods, 65:55, 1983 (2) Jacobs, JP, J. Natl. Cancer Inst., 34; 231, 196
Five The following examples illustrate the invention in more detail. This will enable those skilled in the art to fully understand the present invention. However, the present invention should not be limited to the specific descriptions given in the examples.

Example 1 5- (2-chloro-2-propenyl) -11-ethyl-5,
11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepin-6-one 5- (2-chloro-2-propenyl) -5,11-dihydro-6H-pyrido [2,3- b] [1,4] benzodiazepine
9.7 g (0.034 mol) of 6-one (melting point 157-160 ° C.) was suspended in 100 ml of anhydrous dimethylformamide. The mixture was purged thoroughly with dry nitrogen and 1.8 g (0.0375 mol) of a 50% dispersion of sodium hydride in mineral oil was slowly added in small portions via an inlet protected by a potassium chloride tube. The addition of the first portion of sodium hydride results in a rapidly exothermic reaction, which rapidly raises the temperature of the mixture to about 60 ° C. After the addition of sodium hydride is complete (30-35
Min), the reaction mixture was cooled to room temperature, and then 7.8 g (0.05 mol) of ethyl iodide was added dropwise. Then remove the flask
The reaction mixture was stirred and heated for 2 hours in an oil bath heated to 100 ° C. Meanwhile, it was passed through a device with a gentle flow of dry nitrogen. Subsequent examination by thin-layer chromatography revealed no starting material.

The reaction mixture was cooled to room temperature and 1 ml of methanol was added to destroy any sodium hydride that might still be present. The reaction mixture was evaporated under reduced pressure and the residue was
Dispersed in 0 ml ether and 100 ml water. The organic layer was separated and the aqueous layer was completely extracted with ether. The combined organic layers were dried over sodium sulfide, after which the inorganic salts were removed by filtration under reduced pressure and the filtrate was evaporated to dryness under reduced pressure. The resulting highly viscous red oil was purified by column chromatography on silica gel (0.4 mL) using a chloroform / ethyl acetate eluent with a volume ratio of 4/1 (v / v).
(2-0.5 mm). The crystalline crude product obtained by evaporating the appropriate fractions is recrystallized from cyclohexane and from diethyl ether and 5.9 g of colorless crystals having a melting point of 80-82 ° C. (55% of theory) ) Got.

Example 2 2,5,8,11-Tetramethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one Same as the method described in Example 1. 2,8−
Dimethyl-5,11-dihydro-6H-pyrido [2,3-b]
[1,4] -benzodiazepin-6-one (melting point 204-206
° C) and methyl iodide from 2,5,8,11-tetramethyl-
5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one (mp 139-141 ° C, melting point 100-1
(Recrystallized from ligroin at 40 ° C.). The yield was 78% of theory.

Example 3 5,11-Diethyl-10-methyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one Similar to the method described in Example 1. According to the method, 5-ethyl-10-methyl-5,11-dihydro-6H-pyrido [2,3
-B] [1,4] -benzodiazepin-6-one (mp 168
~ 170 ° C) and ethyl iodide to give 5,11-diethyl-10
-Methyl-5,11-dihydro-6H-pyrido [2,3-b]
[1,4] -benzodiazepin-6-one (melting point 143-144)
Recrystallized from ligroin with a melting point of 100-140 ° C)
Was manufactured. The yield was 54% of the theoretical value.

Example 4 5,8-Dimethyl-11-ethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one Similar to the method described in Example 1. The title compound was obtained by the method.

Example 5 2,5,10,11-Tetramethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one Same as the method described in Example 1. 2,5,10
-Trimethyl-5,11-dihydro-6H-pyrido [2,3-
b] [1,4] -benzodiazepin-6-one (melting point 140-
142 ° C) and methyl iodide from 2,5,10,11-tetramethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4]-
Benzodiazepin-6-one (melting point 131-132 ° C, melting point 10
(Recrystallized from ligroin at 0 to 140 ° C.). The yield was 28% of the theoretical value.

Example 6 [11- [5-11-dihydro-6-oxo-6H-pyrido [2,3-b] [1,4] benzodiazepin-11-yl] acetic acid tert-butyl ester Mechanical stirrer, cooler, equalizer In a round bottom flask equipped with a pressure down funnel, a thermometer, and a glass joint with a rubber septum, place 5-11 in anhydrous tetrahydrofuran 550.
-Dihydro-6-oxo-6H-pyrido [2,3-b] [1,
4] A suspension of 10.5 g (0.05 mol) of benzodiazepin-6-one was added. After cooling to 0 ° C. in an externally applied ice-salt bath, a solution of 1.55 mol of n-butyllithium in hexane
96 (0.149 mol) was added by injecting from a syringe through the septum at a temperature below 10 ° C. After holding the ice bath for 15 minutes, the mixture was heated to 30-35 ° C. to complete the metallization. Thereafter, the mixture was cooled to -10 ° C and bromoacetic acid tert-butyl ester in anhydrous tetrahydrofuran 50.
A stirred mixture of 0 (10.64 g, 0.055 mol) solution was added dropwise. The resulting cloudy reaction mixture was subsequently stirred at -10 ° C for 2 hours and at room temperature for 2 hours. Thereafter, the resulting suspension was poured into the salt solution of 1 with stirring and the resulting mixture was completely extracted with ethyl acetate. The combined organic layers are washed with water, dried over anhydrous sodium sulfate,
Filtered. The solvent is evaporated off under reduced pressure and the residue is then subjected to column chromatography on silica gel (0.063-0.2 mm)
And purified using a mixture of dichloromethane / ethyl acetate / cyclohexane in a volume ratio of 1400/400/50 as eluent. Evaporation of the appropriate fractions gave a colorless resin. This is 11- [5-11-dihydro-6-oxo-6H-
Pyrid- [2,3-b] [1,4] benzodiazepin-11-yl] acetic acid tert-butyl ester was identified.
The yield was 5.6 g (34% of logic).

Example 7 2-chloro-5-ethyl-11-methyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepine-6
-One 2-chloro-11-methyl-5,11-dihydro-6-oxo-6H-pyrido [2,3-b] [1,4] benzodiazepine-
6-one (64.9 g, 0.25 mol) and potassium methoxide 20
g (0.285 mol) was mixed with a mixture consisting of anhydrous dioxane 250 and tert-butanol 250, and the mixture was refluxed for 2 hours. Then, the reaction solution was cooled, and ethyl iodide 45 g
(0.3 mol) was added dropwise over 45 hours, and the resulting mixture was refluxed again for 2 hours. Subsequently, the resulting reaction solution was filtered while hot, the filtrate was evaporated under reduced pressure, and the residue was recrystallized from cyclohexane. 2-chloro-5 with a melting point of 162-164 ° C
-Ethyl-11-methyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepin-6-one 32.4 g
(45% of theory).

Example 8 5,8,11-trimethyl-5,11-dihydro-6H-pyrido [2,
3-b] [1,4] Benzodiazepin-6-one By a method similar to the method described in Example 1, melting point 17
From 5,8-dimethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one and methyl iodide at 6-178 ° C, 5,8,11- Trimethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one (recrystallized from ligroin having a melting point of 147 to 149 ° C and a melting point of 100 to 140 ° C) Manufactured. The yield was 58% of theory.

Example 9 The following known compounds were prepared using the method described in US Pat. No. 3,539,544.

a) 11-methyl-5,11-dihydro-6H-pyrido [2,3
-B] [1,4] -benzodiazepin-6-one b) 11-ethyl-5,11-dihydro-6H-pyrido [2,3
-B] [1,4] -benzodiazepin-6-one c) 5,11-dihydro-6H-pyrido [2,3-b] [1,4]
-Benzodiazepin-6-one d) 5-ethyl 11-methyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one e) 5-n-propyl- 11-methyl-5,11-dihydro-6H-pyrido [2,3, -b] [1,4] -benzodiazepin-6-one f) 5-methyl-11-ethyl5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one g) 5,11-diethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepine- 6-one h) 5- (2-propenyl) -11-ethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one Example A capsule or tablets a-1 component amount (mg) example 4 compound 50 starch 160 microcrystalline cellulose 90 sodium starch glucammonium Tate 10 magnesium stearate 2 Fumed colloidal silica 1 A-2 Ingredients Amount (mg) of the compound of Example 4 of the compound 50 dicalcium phosphate 160 Microcrystalline cellulose 90 Stearic acid 5 Sodium starch glycolate 10 Fumed colloidal silica 1 Example 4 above The excipients shown, excluding the lubricant, are blended into a premixed powder mixture. Thereafter, a lubricant is blended, and the resulting blend is compressed into tablets or filled into hard gelatin capsules.

Example B Amount of Solution Component for Injection Compound of Example 4 500 mg Ethanol 25 Purified Water for Injection Adjust the total amount to 100 Add the compound of Example 4 to ethanol and mix until the solution becomes transparent. Water is added, and the resulting solution is then filtered into a suitable vial or ampoule and sterilized by autoclaving.

Example C Inhalation Solution Component Amount Compound of Example 4 500 mg Propylene glycol 30 Benzalkonium chloride 200 mg EDTA 200 mg Water Make the total amount 100 The excipient was mixed, and then the compound of Example 4 was added.
Continue mixing until the solution is clear. Water is added, then the resulting solution is filtered into a suitable vial or ampoule.

Continued on the front page (72) Inventor Güntel Schmidt Germany Day 8000 München 19 Nünfenburger Strasse 155 (72) Inventor Wolfhard Engel Germany Day 7950 Biberach 1 Mozartstrasse 13 (72) Inventor Volkhard Auster, Germany Day 7950 Biberach 1 Kapellenweg 7 (72) Inventor Carl Dee Hargrave, Connecticut, United States 06805 Brookfield Edna Drive 4 (58) Fields studied (Int. Cl. ⁶ , DB name) A61K 31/415 C07D 471/04

Claims (4)

(57) [Claims] 1. Prevention or treatment of HIV-1 infection, comprising a pharmaceutically acceptable carrier and a prophylactically or therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable acid addition salt of said compound. Pharmaceutical compositions suitable for: (In the formula, Z represents an oxygen atom or a sulfur atom, and R ¹ is a hydrogen atom, an alkyl group or a fluoroalkyl group having 1 to 4 carbon atoms, a cyclopropyl group, an alkenyl group or an alkynyl group having 3 to 4 carbon atoms. , 2
-Halo-propen-1-yl group, arylmethyl group (wherein, the aryl moiety represents a phenyl group or a thienyl group,
These represent unsubstituted or substituted with a methyl group, a methoxy group or a halogen atom), an acetyl group, or an alkoxyalkyl group or an alkylthioalkyl group having 2 to 3 carbon atoms, and R ² represents 1 to 4 carbon atoms. An alkyl group or a fluoroalkyl group, a cycloalkyl group having 3 to 5 carbon atoms, an alkenyl group or an alkynyl group having 2 to 4 carbon atoms, an alkoxyalkyl group or an alkylthioalkyl group having 2 to 3 carbon atoms, 2 to 3 alkanoyl groups, C 2 to C 4 hydroxyalkyl groups, arylmethyl groups (wherein the aryl moiety represents a phenyl group, a thienyl group or a furanyl group, which is unsubstituted or has 1 to 3 carbon atoms) Alkyl group or alkoxy group, hydroxyl group or halogen Phenyl group (unsubstituted or has 1 carbon atom)
Or 3 substituted with an alkyl group or an alkoxy group, a halogen atom or a hydroxyl group) or an alkoxycarbonylmethyl group in which the alkoxy moiety has 1 to 5 carbon atoms, and R ³ , R ⁴ and R ⁵ are each independently Represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, provided that at least one of these substituents represents a hydrogen atom, or one of R ³ , R ⁴ and R ⁵ represents a butyl group, Alkanoyl group having 2 to 4 carbon atoms, alkoxycarbonyl group having 2 to 4 carbon atoms, hydroxyalkyl group having 1 to 4 carbon atoms, alkoxycarbonylalkyl group having alkoxy moiety and alkyl moiety each having 1 to 2 carbon atoms, halogen Atom, trihalomethyl group, hydroxyl group, C1 or C3 alkoxy group, C1 to C3 Alkylthio group, alkanoyloxy group having 2 to 3 carbon atoms, alkanoylamino group having 1 to 3 carbon atoms, aminoalkyl group having 1 to 3 carbon atoms, each alkyl portion containing 1 to 2 carbon atoms Mono- or di-alkylamino group, 2 carbon atoms
To 3 carboxyalkyl groups, cyano groups, nitro groups, carboxy groups, carbamyl groups, amino groups, azido groups, mono- or di-alkylaminoalkyl groups wherein each alkyl moiety has 1 to 2 carbon atoms. Provided that the remaining two substituents represent a hydrogen atom or a methyl group, or when Z represents an oxygen atom, one of R ³ , R ⁴ and R ⁵ has 1 to 3 carbon atoms, an alkylsulfinyl group or Represents an alkylsulfonyl group, provided that the remaining two substituents represent a hydrogen atom or a methyl group, and R ⁶ , R ⁷ , R ⁸ and R ⁹ each represent a hydrogen atom, or R ⁶ , R ⁷ , one of R ⁸ and R ⁹ are alkyl of 1 to 4 carbon atoms, alkanoyl of 2 -C 4, C 2 -C 4 alkoxycarbonyl group, hydroxyalkyl of 1 to 4 carbon atoms A kill group, an alkoxycarbonylalkyl group wherein the alkoxy and alkyl portions each have 1 to 2 carbon atoms, a halogen atom, a trihalomethyl group, a hydroxyl group, an alkoxy group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms. An alkylthio group, an alkanoyloxy group having 2 to 3 carbon atoms, an alkanoylamino group having 1 to 3 carbon atoms, an aminoalkyl group having 1 to 3 carbon atoms,
A mono- or di-alkylamino group having 2 to 2 carbon atoms, a carboxyalkyl group having 2 to 3 carbon atoms, a cyano group, a nitro group, a carboxy group, a carbamyl group, an amino group, an azido group, Represents a mono- or di-alkylaminoalkyl group having 1 to 2 carbon atoms, wherein the remaining three substituents represent a hydrogen atom, or two of the remaining three substituents represent a hydrogen atom, One represents a methyl group, an ethyl group or a halogen atom) 2. In the above formula I, Z represents an oxygen atom or a sulfur atom, and R ¹ represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, an allyl group, a propargyl group, a 2-halopropen-1-yl. R ² is an alkyl group having 1 to 4 carbon atoms, 3 carbon atoms.
A cycloalkyl group having 2 to 4 carbon atoms, an alkenyl group or an alkynyl group having 2 to 4 carbon atoms, an alkoxyalkyl group or an alkylthioalkyl group having 2 to 3 carbon atoms, an alkanoyl group having 2 to 3 carbon atoms, A hydroxyalkyl group, an arylmethyl group (wherein, the aryl moiety represents a phenyl group or a thienyl group, which is unsubstituted or substituted with a methyl group, a methoxy group or a halogen atom) or an alkoxy moiety of 1 to 4; R ³ , R ⁴ and R ⁵ each independently represent a hydrogen atom or a methyl group, provided that at least one of these substituents represents a hydrogen atom, or R ⁵ is an ethyl group, a propyl group or a butyl group, and the remaining two substituents represent a hydrogen atom or a R ⁶ Atom, a methyl group or an ethyl group, provided that when R ⁷ represents a hydrogen atom or a methyl group, an alkyl group having R ⁷ is C 1 -C 2, an acetyl group,
A hydroxyalkyl group having 1 to 2 carbon atoms, an alkoxycarbonyl group having 2 to 3 carbon atoms, an alkoxycarbonylalkyl group having an alkoxy portion and an alkyl portion each having 1 to 2 carbon atoms, a halogen atom, a trifluoromethyl group , A hydroxyl group, an alkoxy group having 1 to 2 carbon atoms, an alkylthio group having 1 to 2 carbon atoms, an acetyloxy group, an alkanoylamino group or a cyano group having 1 to 2 carbon atoms, provided that R ⁸ is hydrogen. Represents an atom: R ⁸ is an alkyl group having 1 to 2 carbon atoms, an acetyl group,
A hydroxylalkyl group having 1 to 2 carbon atoms, an alkoxycarbonyl group having 2 to 3 carbon atoms, an alkoxycarbonylalkyl group having an alkoxy moiety and an alkyl moiety each having 1 to 2 carbon atoms, a halogen atom, a trifluoromethyl group , A hydroxyl group, an alkoxy group having 1 to 2 carbon atoms, an alkylthio group having 1 to 2 carbon atoms, an alkanoylamino group or a cyano group having 1 to 2 carbon atoms, provided that R ⁷ represents a hydrogen atom Or R ⁷ and R ⁸ both represent a hydrogen atom, a methyl group or a halogen atom, and R ⁹ represents a hydrogen atom or a methyl group, provided that R ⁸
The composition according to claim 1, wherein represents a hydrogen atom or a methyl group. In the above formula I, Z represents an oxygen atom or a sulfur atom, R ¹ is a hydrogen atom, a 2-halo-2-propen-1-yl group,
Or an alkyl group having 1 to 3 carbon atoms, R ² represents an alkyl group having 1 to 4 carbon atoms or a cycloalkyl group having 3 to 4 carbon atoms, and R ^{3 to} R ⁹ each represent a hydrogen atom The composition according to claim 1. 4. A compound selected from the following group and a pharmacologically acceptable acid addition salt of the compound. a) 5- (2-chloro-2-propenyl) -11-ethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] -benzodiazepin-6-one; b) 2, 5,8,11-tetramethyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepin-6-one; c) 5,11-diethyl-10-methyl-5, 11-dihydro-6H
-Pyrido [2,3-b] [1,4] benzodiazepin-6-one; d) 5,8-dimethyl-11-ethyl-5,11-dihydro-6H
-Pyrido [2,3-b] [1,4] benzodiazepin-6-one; e) 2,5,10,11-tetramethyl-5,11-dihydro-6H-
Pyrido [2,3-b] [1,4] benzodiazepin-6-one; f) [11- [5,11-dihydro-6-oxo-6H-pyrido [2,3-b] [1,4] Benzodiazepin-11-yl]]-
Tert-butyl acetate; g) 2-chloro-5-ethyl-11-methyl-5,11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepin-6-one; and h) 5,8,11-Trimethyl-5,11-dihydro-6H-pyrido1 [2,3-b] [1,4] benzodiazepin-6-one.